Refolding of a paramyxovirus F protein from prefusion to postfusion conformations observed by liposome binding and electron microscopy

Abstract

For paramyxoviruses, two viral glycoproteins are key to the entry process: an attachment protein (HN, H, or G) and the fusion protein (F). The F protein folds to a metastable state that can be triggered to undergo large conformational rearrangements to a fusogenic intermediate and a more stable postfusion state. The triggering mechanism that controls paramyxovirus fusion has not been elucidated. To correlate the molecular structure of a soluble form of the prefusion F (PIV5 F-GCNt) with the biological function of F, soluble F protein was triggered to refold. In the absence of HN, heat was found to function as a surrogate F trigger, and F associated with liposomes and aggregated on sucrose density gradients. Electron microscopy data showed that triggered F formed rosettes. Taken together these data suggest that release and membrane insertion of the hydrophobic fusion peptide require both cleavage of F and heat. Heating of cleaved F causes conversion to a postfusion form as judged by its ''golf tee'' morphology in the electron microscope. Heating of uncleaved F also causes conversion of F to a morphologically similar form. The reactivity of the F protein with conformation-specific mAbs and peptide binding suggest that soluble F-GCNt and membrane-bound F proteins refold through a comparable pathway.conformational change ͉ fusion peptide ͉ metastable ͉ trigger

Biochemical, Conformational, and Immunogenic Analysis of Soluble Trimeric Forms of Henipavirus Fusion Glycoproteins

“…It was shown by Yin et al (72,73) that appending the GCNt helix to the HRB of a truncated PIV5 sF construct yielded a prefusion structure in comparison to the structure for the non-GCNt-appended hPIV3 sF glycoprotein, which spontaneously folds to a postfusion configuration. In addition, it was demonstrated that the prefusion PIV5 sF GCNt could be activated and refolded to a postfusion conformation in vitro by heat and trypsin treatment (23). These earlier findings, together with the data described above, suggested that the HeV and NiV sF GCNt glycoproteins are also likely folded into a prefusion conformation, whereas the non-GCNt-appended forms are not and likely have a configuration similar to a postfusion configuration.…”

“…Paramyxovirus F glycoproteins demonstrate a propensity to aggregate in solution or when extracted out of membranes, which may be the result of an exposed Fp (23,48). The alteration of hydrophobic residues within the Fp of RSV F yielded an sF construct with a reduced tendency to aggregate (48).…”

“…The prefusion sF GCNt particles adopted primarily two preferred orientations on the carbon support of the EM grid, displaying a top view of the trimer head region and a side view of the spherical head region with a thin stalk domain similar to the lollipop structure previously observed for the prefusion F of PIV5 (23), Newcastle disease virus (NDV) (65), and human metapneumovirus (hMPV) (68). The particles with the postfusion conformation of the non-GCNt-appended NiV sF dFp adopted a single preferred orientation revealing a side view of the trimer head region and stalk domain, with the head region being distinctly less round, more box shaped, and similar to the golf tee shape of postfusion PIV5, NDV, and hMPV (23,65,68).…”

“…These earlier findings, together with the data described above, suggested that the HeV and NiV sF GCNt glycoproteins are also likely folded into a prefusion conformation, whereas the non-GCNt-appended forms are not and likely have a configuration similar to a postfusion configuration. To examine this possibility, the purified sF GCNt , presumably in a prefusion conformation, was activated and triggered using techniques similar to those reported by Connolly et al (23). Because sF GCNt could be properly cleaved in vitro by trypsin cleavage, shown earlier, we could examine whether the HeV and NiV sF GCNt could be cleaved and triggered to fold into a postfusion configuration by applying heat (50°C, 15 min) and trypsin.…”

Efficient replication of a paramyxovirus independent of full zippering of the fusion protein six-helix bundle domain

“…The F trypsin mutant meets these conditions, based on our F-protein coimmunoprecipitation data and the previous observation that MeV F trypsin is fully reactivatable by the addition of exogenous trypsin protease (38). Furthermore, uncleaved F trimers derived from the related paramyxovirus PIV5 were shown to remain competent in refolding into a postfusion conformation (19,52), confirming that no obvious structural constraints prevent the conformational rearrangement of F trypsin -containing trimers in a dominant-negative manner.…”